The present invention claims priority to the following applications: 1) EP 96 118 490.0, filed Nov. 19, 1996; 2) EP 97 101 085.5, filed Jan. 24, 1997; and 3) PCT/EP97/06002, filed Oct. 30, 1997.
The invention concerns a recombinant collagenase type I (CHC I) from Clostridium histolyticum and its use for isolating cells and groups of cells.
Bacterial collagenases, e.g. from Clostridium histolyticum, are used to digest tissues and to isolate individual cells or groups of cells (e.g. islets) (islets: Sutton et al., Transplantation 42 (1986) 689-691; liver: Quibel et al., Anal. Biochem. 154 (1986) 26-28; bones: Hefley et al., J. Bone Mineral Res. 2 (1987) 505-516; umbilical cord: Holzinger et al., Immunol. Lett. 35 (1993) 109-118). Two different collagenase types are known from Clostridium histolyticum (M. F. French et al., J. Protein Chemistry 11 (1992) 83-97). The isolation and purification of collagenases from Clostridium histolyticum is described for example in E. L. Angleton and H. E. van Wart, Biochemistry 27 (1988) 7413-7418 and 7406-7412.
Collagenases I were isolated and described by M. D. Bond and H. E. Van Wart, Biochemistry 23 (1984) 3077-3085 and 3085-3091 as well as by H. E. Van Wart, Biochemistry 24 (1985) 6520-6526. Accordingly the collagenases a (MW 68 kD), xcex2 (MW 115 kD), xcex3 (MW 79 kD) and xcex7 (MW 130 kD) are known as collagenases I. Collagenases I and collagenases II differ in their relative activity towards collagen and towards synthetic peptides. Collagenases I have higher activities towards collagen and gelatin and lower activities towards the short-chained peptides than collagenase II (Bond and Wart, Biochemistry 23 (1984) 3085-3091).
Further collagenases are described in the U.S. Pat. Nos. 5,418,157 and 5,177,011 with a molecular weight of 68 kD. WO 91/14447 also describes a collagenase with a molecular weight of 68 kD. A recombinant collagenase with a molecular weight of ca. 110 kD is described in WO 94/00580 and a sequence is stated for it. Nothing is stated in WO 94/00580 about its specificity and in particular whether it is a collagenase I or II. In addition to a collagenase with a molecular weight of 110 kD, a 125 kD collagenase is additionally mentioned which it is claimed can also be produced recombinantly. However, WO 94/00580 does not give more details about this collagenase.
The object of the present invention is to provide a highly active stable collagenase class I from Clostridium histolyticum (CHC I).
The object is achieved by a process for the production of a polypeptide which has the properties of a CHC I from Clostridium histolyticum, has a given amino acid composition and is obtainable by expression of an exogenous nucleic acid in prokaryotic or eukaryotic host cells and isolation of the desired polypeptide wherein the nucleic acid codes for a polypeptide having sequence ID NO:2 or a polypeptide which is extended N-terminally by one or several amino acids having the sequence ID NO:3.
It has surprisingly turned out that a CHC I according to the invention has a high collagenase class I activity and is very stable. A CHC I having the amino acid sequence SEQ ID NO:2 and a CHC I which is N-terminally extended by one or several amino acids having SEQ ID NO:3 are particularly preferred.
The CHC I according to the invention is a highly pure enzyme which can be produced in large amounts. The CHC I according to the invention is not contaminated by other clostridial enzymes and is free of toxins.
The CHC I according to the invention is especially suitable for isolating cells from tissues of mammals, preferably from human tissue, for an application in cell therapy (transplantation, immunotherapy) and for an application in gene therapy in tissues: e.g. pancreas, liver, bone, cartilage, skin, brain and nerve tissue, fat, muscle, heart, endothelium, kidney, solid tumours and for the purification of ulcers.
In addition this highly pure enzyme (preferably in a mixture with other-highly pure enzymes (such as collagenase II and neutral protease)) is particularly suitable for the isolation of cells whose surface molecules/markers (antigens) should not be changed. Preferred applications of this are for example the dissociation of solid tumours of all types in vitro (e.g. colon, breast etc.) for adoptive immunotherapy and for general diagnosis such as e.g. to detect rare cells. from tissues and solid tumours by means of specific surface markers/molecules.
The production of the recombinant CHC I can be carried out according to methods familiar to a person skilled in the art.
For this a DNA molecule is firstly produced which is capable of producing a protein which has the activity of CHC I. The DNA sequence is cloned into a vector which can be transferred into a host cell and can be replicated there. Such a vector contains promoter/operator elements which are necessary to express the DNA sequences in addition to the CHC I sequence. This vector which contains the CHC I sequence and the promoter/operator elements is transferred into a host cell which is able to express the DNA of CHC I. The host cell is cultured under conditions which are suitable for the amplification of the vector and CHC I is isolated. In this process suitable measures ensure that the protein can adopt an active tertiary structure in which it exhibits CHC I properties.
The nucleic acid sequence and protein sequence can be modified to the usual extent. Such modifications are for example:
Modification of the nucleic acid in order to introduce various recognition sequences of restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis.
Modification of the nucleic acid to incorporate preferred codons for the host cell.
Extension of the nucleic acid by additional operator elements in order to optimize the expression in the host cell.
Substitution or deletion of amino acids while retaining the basicity and the spatial structure of CHC I. It is advantageous to preserve 85% or more and preferably 90% or more of the original amino acid sequence.
A further subject matter of the invention is a polypeptide with the properties of a collagenase class I from Clostridium histolyticum with the amino acid sequence according to SEQ ID NO:2 or a polypeptide extended N-terminally by one or several amino acids of SEQ ID NO:3. A further subject matter of the invention is a nucleic acid coding for such a protein.
The protein is preferably produced recombinantly in microorganisms, in particular in prokaryotes and in this case in E. coli. 
Suitable expression vectors must contain a promoter which allows expression of the protein in the host organism. Such promoters are known to a person skilled in the art and are for example the lac promoter (Chang et al., Nature 198 (1977) 1056), trp promoter (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), xcexPL promoter (Shimatake et al., Nature 292 (1981) 128) and T5 promoter (U.S. Pat. No. 4,689,406). Synthetic promoters are also suitable such as for example the tac promoter (U.S. Pat. No. 4,551,433). Coupled promoter systems are also suitable such as the T7-RNA polymerase/promoter system (Studier et al., J. Mol. Biol. 189 (1986) 113). Hybrid promoters composed of a bacteriophage promoter and the operator region of the microorganism (EP-A 0 267 851) are equally suitable. An effective ribosome binding site is necessary in addition to the promoter. In the case of E. coli this ribosome binding site is denoted the Shine-Dalgarno (SD) sequence (Sambrook et al., xe2x80x9cExpression of cloned genes in E. colixe2x80x9d in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA).
In order to improve the expression it is possible to express the protein as a fusion protein. In this case a DNA sequence which codes for the N-terminal part of an endogenous bacterial protein or for another stable protein is usually fused to the 5xe2x80x2 end of the DNA coding for the CHC I. Examples of this are for example lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185 (1990) 161-166).
After expression of the vector, preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably cleaved with enzymes (e.g. factor Xa) (Nagai et al., Nature 309 (1984) 810). Other examples of cleavage sites are the IgA protease cleavage site (WO 91/11520, EP-A 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio/Technology 7 (1989) 698).
The proteins expressed in this manner in bacteria are isolated in the usual way by lysing the bacteria and protein isolation.
In a further embodiment it is possible to secrete the proteins as active proteins from the microorganisms. For this a fusion product is preferably used which is composed of a signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid which codes for the protein. In this process the protein is either secreted into the medium (in the case of gram-positive bacteria) or into the periplasmic space (in the case of gram-negative bacteria). It is expedient to place a cleavage site between the signal sequence and the sequence coding for CHC I which enables cleavage of the protein either during processing or in an additional step. Such signal sequences are derived for example from ompA (Ghrayeb et al. EMBO J. 3 (1984) 2437) or phoA (Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212).
The vectors in addition also contain terminators. Terminators are DNA sequences which signal the end of a transcription process. They are usually characterized by two structural features: an inverse repetitive G/C-rich region which can intramolecularly form a double helix and a number of U(or T) residues. Examples are the main terminator in the DNA of the phage fd (Beck and Zink, Gene 16 (1981) 35-58) and rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107-127).
In addition the expression vectors usually contain a selectable marker in order to select the transformed cells. Such selectable markers are for example the resistance genes for ampicillin, chloroamphenicol, erythromycin, kanamycin, neomycin and tetracyclin (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469). Selectable markers which are also suitable are the genes for substances that are essential for the biosynthesis of substances necessary for the cell such as e.g. histidine, tryptophan and leucine.
Numerous suitable bacterial vectors are known. Vectors have for example been described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Natl. Acad. Sci. USA 79 (1982) 5582), E. coli (Aman et al., Gene 40 (1985) 183; Studier et al., J. Mol. Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Appl. Environ. Microbiol. 54 (1988) 655), Streptococcus lividans and Streptomyces lividans (U.S. Pat. No. 4,747,056).
Further genetic engineering methods for the construction and expression of suitable vectors are described in J. Sambrook et al., Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, N.Y.
Apart from in prokaryotic microorganisms, recombinant CHC I can also be expressed in eukaryotes (such as for example CHO cells, yeast or insect cells). The yeast system or insect cells is preferred as the eukaryotic expression system. Expression in yeast can be achieved by means of three types of yeast vectors (integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors. More details of this are for example described in S. M. Kingsman et al., Tibtech 5 (1987) 53-57).
The invention in addition concerns a process for disintegrating cell tissue and releasing cells or groups of cells contained therein by incubating the cell tissue with a collagenase class I from Clostridium histolyticum until the cells or groups of cells have been released to the desired extent and separating the cells or groups of cells from the cell tissue fractions. The separation of the cells or groups of cells from the cell tissue fractions is preferably carried out by centrifugation using a density gradient.
Cells or groups of cells are usually isolated from tissues (e.g. pancreas, liver, skin, endothelium, umbilical cord, bone, tumour tissue) by incubating organs, parts of organs or tissues with enzymes which dissolve the surrounding extracellular connective tissue matrix (islets: Sutton et al., Transplantation 42 (1986) 689-691; liver: Quibel et al., Anal. Biochem. 154 (1986) 26-28; bone: Hefley et al., J. Bone Mineral Res. 2 (1987) 505-516); umbilical cord: Holzinger et al., Immunol. Lett. 35 (1993) 109-118). Tumour cells isolated in this manner can be used advantageously for tumour vaccination and/or adoptive immunotherapy. Tissue disintegration can also be carried out by perfusing the entire organ (Ricordi et al., Diabetes 37 (1988) 413-420) with an enzyme solution. Important factors in this process, in addition to the composition of the enzyme mixture, are the duration, the pH value and the temperature of the digestion as well as the mechanical action e.g. by shaking and addition of metal balls. Since extracellular connective tissue matrix often has a high proportion of collagen, collagenases play a special role (Wolters, Hormone and Metabolic Research 26 (1994), 80).
The process according to the invention is preferably used to isolate islets or islet cells from pancreatic tissue.
In addition, the addition of further enzymes such as proteinases (e.g. neutral protease, cf. example 5 or other metalloproteases; serine proteases such as trpysin, chymotrypsin, plasmin etc.; cysteine proteases; aspartate proteases), elastases, hyaluronidase, lipases or other collagenases may be advantageous for the quality of the digestion.